The ‘Culicoides obsoletus group’ in Italy: relative abundance, geographic range, and role as vector for Bluetongue virus Maria Goffredo1*, Rudy Meiswinkel2, Valentina Federici1, Francesca Di Nicola1, Giuseppe Mancini1, Carla Ippoliti1, Alessio Di Lorenzo1, Michela Quaglia1, Adriana Santilli1, Annamaria Conte1 and Giovanni Savini1

1 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise‘ G. Caporale’, Teramo, Italy. 2 Entomologist, Santa Maria del Monte, Via Pretarone 14, Rocca di Cave, 00030 Rome, Italy. * Corresponding author at: Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise‘ G. Caporale’, Campo Boario, 64100 Teramo, Italy. Tel.: +39 0861 332416, e-mail: [email protected].

Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1 Accepted: 03.06.2016 | Available on line: 30.09.2016

IV International Conference on Bluetongue and Related Orbiviruses. November 5‑7, 2014 ‑ Rome, Italy ‑ Selected papers

Keywords Summary Avaritia, As Bluetongue virus (BTV) spread in Italy following its first incursion in 2000, it soon became Culicoides chiopterus, apparent that, besides Culicoides imicola, additional species of the subgenus Avaritia Culicoides dewulfi, were involved as vectors, namely one or more of the species that belong to the so-called Obsoletus complex, ‘Culicoides obsoletus group’, which comprises C. dewulfi, C. chiopterus, C. obsoletus sensu Vector competence. stricto, C. scoticus and C. montanus; the three last named species are considered generally as forming the Obsoletus complex. This study presents the findings made over the last decade and more, within the Italian entomological surveillance program for Bluetongue. It describes the integrated morphological and molecular approach used to identify the species of the ‘C. obsoletus group’, maps in detail their relative abundances and geographic ranges in Italy, clarifies the hitherto unknown comparative seasonal abundances of C. obsoletus s.s. and C. scoticus in a site in Central Italy, and provides further details on the potential vector status of five species of the ‘C. obsoletus group’, with emphasis on C. obsoletus s.s., C. scoticus and C. montanus. Unlike the situation in Northern Europe, Culicoides dewulfi and C. chiopterus are uncommon to rare in Italy. In contrast, the Obsoletus complex occurs abundantly throughout Italy, with C. obsoletus s.s. being the most prevalent and ecologically adaptive of the three species making up the complex. A longitudinal study conducted at a site in Central Italy revealed that: (i) species of the Obsoletus complex prefer horses to sheep; (ii) their parity rates range from 10% (March) to 56% (November); (iii) throughout the year C. scoticus is consistently more abundant than C. obsoletus s.s.; (iv) abundances in both, C. obsoletus s.s. and C. scoticus, peak in May-June, with the peak of the latter species being more evident. Bluetongue virus was first isolated from wild caught midges of the Obsoletus complex in 2002. Thereafter, pools of selected parous midges collected across Italy, and during multiple outbreaks of BT, have been found consistently PCR-positive for the virus. More recently, viral RNA has been detected in field specimens ofC. dewulfi, C. obsoletus s.s., C. scoticus and C. montanus.

Il “Culicoides obsoletus group” in Italia: abbondanza, distribuzione geografica e ruolo vettoriale

Parole chiave Riassunto Avaritia, Sin dalle prime incursioni del virus della Bluetongue (BTV) in Italia a partire dal 2000, è Competenza vettoriale, risultato evidente che altre specie di vettori del sottogenere Avaritia fossero coinvolte, Culicoides chiopterus, oltre a Culicoides imicola, in particolare quelle del cosiddetto “Culicoides obsoletus group”, Culicoides dewulfi, che comprende C. dewulfi, C. chiopterus, C. obsoletus sensu stricto, C. scoticus e C. montanus; Obsoletus complex. le ultime tre specie formano nell’insieme l’Obsoletus complex. Questo lavoro riassume i risultati ottenuti in oltre dieci anni di sorveglianza entomologica per la BT. Descrive il metodo di identificazione di specie, basato su un approccio integrato morfologico e molecolare, riporta le mappe di distribuzione e abbondanza delle singole specie, nonché l’abbondanza relativa stagionale di C. obsoletus s.s. e C. scoticus in un sito di cattura permanente nell’Italia

235 ‘C. obsoletus group’ in Italy Goffredo et al.

Centrale, e fornisce informazioni sul possibile ruolo vettoriale in Italia di cinque specie del “C. obsoletus group”. Al contrario di quanto avviene in Nord Europa, Culicoides dewulfi e C. chiopterus non sono specie comuni in Italia. Invece l’Obsoletus complex è abbondante in tutta la penisola, soprattutto C. obsoletus s.s., che sembra infatti essere la specie più adattabile. Uno studio longitudinale, condotto in un sito permanente di cattura nell’Italia centrale, ha evidenziato che: (i) le specie dell’Obsoletus complex preferiscono i cavalli alle pecore; (ii) la percentuale dei parous nella popolazione varia dal 10% (marzo) al 56% (novembre); (iii); C. scoticus è costantemente più abbondante di C. obsoletus s.s., durante tutto l’anno; (iv); entrambi C. obsoletus s.s. e C. scoticus mostrano un picco di abbondanza in maggio-giugno, il secondo con un picco più marcato. Il BTV è stato isolato per la prima volta in Italia dall’Obsoletus complex nel 2002, mentre successivamente vari sierotipi sono stati rilevati tramite PCR, nel corso delle diverse epidemie di BT, recentemente, RNA virale è stato riscontrato in campo, per la prima volta in Italia, da C. dewulfi, e inoltre da pool di C. obsoletus s.s., C. scoticus e C. montanus, identificati a livello specifico.

Introduction the phylogeny of the subgenus Avaritia will be resolved only once the entire Holarctic fauna (of Historically, the year 2000 marks the first incursion approximately 30 species) is treated as a whole. of Bluetongue (BT) into Italy. The disease emerged in sheep and spread through the principal Afro‑Asian In the interim, we will continue to use the vector Culicoides imicola Kieffer on Sardinia island. paraphyletic taxon ‘C. obsoletus group’, but retain it In 2001, Bluetongue reached the mainland and in double quotes to signpost its cladistic artificiality. soon began to spread into areas where C. imicola In Europe today, the ‘C. obsoletus group’ (excluding was absent; in time this led to the discovery that C. imicola) comprises seven species; the actual also other biting midge species act as vectors for number is probably closer to ten. As noted above, Bluetongue virus (BTV), namely Culicoides obsoletus five species of the ‘C. obsoletus group’ occur in (Meigen) and Culicoides scoticus Downes and Kettle Italy, namely C. obsoletus sensu stricto, C. scoticus, (Savini et al. 2005). In the decade since its arrival C. montanus, C. dewulfi and C. chiopterus. Integrated in the Mediterranean region, BTV has continued morphological and molecular studies indicate the to incur and spread ever northwards, eventually occurrence of a fourth species of the Obsoletus reaching 58°N, more than 1,000 km beyond its complex in Italy and referred to provisionally as traditional limit of 45°N. Simultaneously, other biting ‘Culicoides sp. unidentified’ (Gomulski et al. 2005); it midge species have been added to the growing does not form part of this study. vector list and now includes also Culicoides dewulfi Below, with reference to the ‘C. obsoletus group’, we Goetghebuer, Culicoides chiopterus (Meigen) and present a brief five‑part review of the entomological Culicoides montanus Shakirzjanova. findings made in Italy since 2000 and obtained under All six of the abovementioned species belong to the the aegis of the Italian entomological surveillance subgenus Avaritia Fox; excepting for C. imicola, most program for Bluetongue: researchers consider the five remaining species i. brief overview (above) of the unresolved to form the ‘C. obsoletus group’. Phylogenetically taxonomy of the ‘C. obsoletus group’; speaking, this group does not form a natural (monophyletic) group so, cladistically speaking, ii. description of the integrated morphological is artificial. Based on adult morphology, only and molecular approach used within the C. obsoletus, C. scoticus and C. montanus form a Italian entomological surveillance program natural group, the Obsoletus complex (not to be of Bluetongue to identify species of the confused with the ‘C. obsoletus group’); this narrow ‘C. obsoletus group’; interpretation has the effect of leavingC. chiopterus iii. mapping of the relative abundances and and C. dewulfi as two separate, free‑floating geographic ranges of each species in Italy; monotypic entities within Avaritia. To a great iv. clarifying the hitherto unknown comparative extent, this fragmented arrangement is supported molecularly, but it remains unclear as to whether seasonal abundances of C. obsoletus s.s. and C. chiopterus should be included (Schwenkenbacher C. scoticus based on a longitudinal study et al. 2009, Schulz et al. 2015) or excluded conducted at a site in Central Italy, and (Meiswinkel et al. 2004, Gomulski et al. 2005, Ander v. the potential vector status of each of the five et al. 2013) from the Obsoletus complex. Ultimately, species of the ‘C. obsoletus group’ is detailed,

236 Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1 Goffredoet al. ‘C. obsoletus group’ in Italy

with emphasis on C. obsoletus s.s., C. scoticus Culicoides species, and utilizes in combination four and C. montanus. wing pattern characters (Goffredo and Meiswinkel, 2004), which must be applied strictly: • distal part of second radial wing cell (r2) Integrated morphological and partially pale; molecular approach used to identify • wing macrotrichia sparse, restricted to distal species of the ‘C. obsoletus group’ half or third of wing; Various approaches are used to identify individual • pale wing spots mostly diffuse, wing bisected species of the C. obsoletus group. These include into a paler basal half and a darker distal half; traditional morphology, advanced morphometrics, pale spot in the apex of r5 poorly defined, mass spectrometry, various polymerase chain round or square, and reaction (PCR) tests, microarrays, and the molecular • absence of a central dark spot in the cubital sequencing of specific gene regions, in particular wing cell. CO1 (barcoding), ITS1 and ITS2. Within the Italian surveillance programme for Bluetongue, the The females of C. chiopterus and C. dewulfi can be laboratory analysis of an entomological field sample distinguished from each other, and from species is treated as rigorously as other kinds of field samples of the Obsoletus complex, based on morphology are (and involving either blood, serum, or animal (Campbell and Pelham‑Clinton 1960, Delécolle tissue). Furthermore, vector species identification, 1985). However, it is far more difficult to identify which requires the screening of hundreds of the females of the three (or more) sibling species samples monthly, needs to be achieved rapidly of the Obsoletus complex and is the reason why and accurately. For these reasons, a standardized species‑specific molecular assays have been protocol, integrating both morphological and developed over the last decade and are being used molecular methods, has been developed, not only increasingly. to differentiate between cryptic species such as Morphologically, C. dewulfi is the only species of C. obsoletus and C. scoticus, but also to confirm Avaritia in Europe in which the female has > 8 long the identity of rarer species (such as C. dewulfi and setae projecting laterally from the first abdominal C. chiopterus) (Figure 1). segment; all the remaining species possess fewer The first step, conducted under the dissecting setae, usually in the range of 2‑5. Furthermore, in microscope, involves the morphological separation C. dewulfi, the two spermathecae differ significantly of specimens of the ‘C. obsoletus group’ from other in size, while the wing has a more rounded shape;

‘Culicoides obsoletus group’ C. chiopterus C. chiopterus, C. dewul , Obsoletus Complex Obsoletus complex  Very pale wing (C. obsoletus, C. scoticus, C. montanus)  Hairy eyes  Multiplex RT-PCR based on ITS-2  Smaller size  Possible con rmation multiplex RT-PCR for the identi cation of C. obsoletus (O), C. scoticus (S) and C. montanus (M) by RT-PCR based on ITS-1 C. scoticus C. obsoletus C. montanus (positive control) (positive control) (positive control) (positive

400 bp 252 bp 213 bp C. dewul  Shape of pale spot in the apex of r5  Asymmetric spermathecae 89 bp  8 to 12 long lateral hairs on the rst O M S O O S abdominal segment RT-PCR for the identi cation of C. dewul  Possible con rmation by RT -PCR based on ITS2 C. dewul (positive control) (positive

294 bp

Figure 1. Integrated morphological and molecular approach to identify the species of the ‘C. obsoletus group’.

Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1 237 ‘C. obsoletus group’ in Italy Goffredo et al.

unfortunately, both characters are very difficult C. obsoletus s.s., C. scoticus and C. montanus; it is based to observe and can be quantify only under a on the internal transcribed spacer 2 (ITS2) (Gomulski dissecting microscope with very high resolution. et al. 2005). An additional species‑specific RT‑PCR, Culicoides chiopterus is one of the smallest European based also on ITS2, was developed to confirm the Culicoides species, however size is not definitive as identification ofC. dewulfi. Control plasmids are larger specimens do exist and will form part of any used as positive reaction controls. These PCRs are collection made. Uniquely, the wing in C. chiopterus is based on field samples of C. obsoletus s.s. (male), yellowish rather than greyish; furthermore, the pale C. scoticus (male), C. dewulfi and C. montanus, spots are very poorly defined and difficult to observe, their identities based on homologous sequences whilst on the wing the macrotrichia are sparse and available in GenBank; to confirm the identification restricted to the distal third. However, the near lack of of C. chiopterus, an RT‑PCR based on ITS1, is used a well‑defined wing pattern means thatC. chiopterus (Mathieu et al. 2007). may be misidentified as a plain‑wing species. However, the 10 plain‑winged species that make up It is important to conclude by saying that within the the European fauna, all differ from C. chiopterus in Holarctic region, the taxonomy of the ‘C. obsoletus having the wing noticeably more ‘hairy’, i.e. the entire group’ is not resolved. At least three cryptic species wing is covered densely with macrotrichia and, in – related closely to the five listed above and not most species, are dark and easy to observe. forming part of current molecular assays – are known to exist (Gomulski et al. 2005, Meiswinkel A practiced eye is however needed to identify et al. 2004, Meiswinkel et al. 2015, Kirkeby et al. 2014, accurately species of Avaritia based on morphology Ander et al. 2012). alone, especially in the female, the sex that forms the bulk (> 95%) of any light‑trap collection made. Especially time‑consuming, is to morphologically differentiate between C. obsoletus s.s. and C. scoticus Distribution and abundance and which occur widely and abundantly across Italy. Based on > 6,000 light‑trap collections made across Consequently, while assaying Culicoides for the Italy, the geographic range and seasonal abundances presence of virus, it is becoming the norm to identify of the species that comprise the ‘C. obsoletus group’ them simultaneously to species molecularly. have been clarified; the three species that comprise Within the protocol used for the Italian BT the Obsoletus complex (C. obsoletus s.s., C. scoticus entomological surveillance, an in‑house multiplex and C. montanus) are mapped separately, based on real time polymerase chain reaction (RT‑PCR) 3,020 individual midges – selected randomly and has been developed to differentiate between identified molecularly – from 87 sites (Figure 2).

A B

Regions Species (%) Obsoletus complex Obsoletus s.s. 1 - 9 C. scoticus 10 - 99 C. montanus 100 - 999 Regions 1000 - 43362

Figure 2. Abundance and distribution map of the Obsoletus complex (A), and relative abundance of C. obsoletus, C. scoticus and C. montanus in Italy (B).

238 Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1 Goffredoet al. ‘C. obsoletus group’ in Italy

Regions Culicoides dewul Regions 0 1 - 10 Culicoides chiopterus 11 - 50 0 51 - 150 1 - 9 151 - 1890 10 - 102

Figure 4. Abundance and distribution map of C. chiopterus in Italy.

Figure 3. Abundance and distribution map of C. dewulfi in Italy. A longitudinal light‑trap study conducted at a permanent collection site in Central Italy revealed Culicoides chiopterus is rarely captured in Italy, with that: C. dewulfi uncommon. Although C. dewulfi occurs i. species of the Obsoletus complex prefer throughout the length of the mainland, it rarely horses to sheep (100 paired collections, exceeds 3% of any light‑trap collection made near U‑Mann Whitney test = 43.47, p < 0.001); livestock; the record catch is 1,890 specimens (relative abundance 21,7%) (Figures 3 and 4). In contrast, the ii. based on 341 collections made near to horses Obsoletus complex, in particular C. obsoletus s.s. between April 2003 and March 2007, adult and C. scoticus, occurs abundantly around livestock females were active throughout the year, their and throughout the whole of Italy. In Northern parity rates ranging from 10% (March) to 56% Italy, C. obsoletus s.s. is the most prevalent, by far. In (November) (Figure 5); sharp contrast, C. montanus – the third species of the iii. based on at least 20 nulliparous females Obsoletus complex – is very rare (Goffredoet al. 2013). (identified by multiplex PCR) extracted at Compared to C. imicola, the Obsoletus Complex random from the 15 largest collections made favours more densely vegetated habitats with monthly between January 2006-March 2007, it increased green leaf density (Conte et al. 2007). was determined that the relative abundances Within the Obsoletus complex, although both C. obsoletus s.s. and C. scoticus are usually present at a collection site, the former is the most prevalent 180000 and ecologically adaptive of the three species that 160000

s 140000 e

make up the Obsoletus complex. g 120000 d i 100000 m

A statistical Bayesian approach (Beta distribution) . 80000 o

was used to assign the sites to a prevalent species N 60000 and resulted in 52 sites for C. obsoletus s.s., eight for 40000 20000 C. scoticus and three for C. montanus. Landscape 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec and remote sensing analysis, applied to these sites (25.1) (19.7) (9.6) (27.0) (29.3) (46.3) (42.8) (39.0) (35.8) (32.3) (55.6) (31.5) Month (Ippoliti et al. 2016) showed that compared to (parity rate %) C. obsoletus s.s., which is often encountered within Nulliparous Engorged Parous Total Obsoletus complex urban and cultivated areas, C. scoticus is more habitat selective, favouring naturally vegetated Figure 5. Age grading and parity rate of the Obsoletus complex in areas, including forests. Finally, C. montanus is by far a permanent collection site in Central Italy. Total number of midges the rarest of the three species and is found mainly per month has been considered, from 341 collections near horses, at lower altitudes in the drier southern half of Italy. between April 2003 and March 2007.

Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1 239 ‘C. obsoletus group’ in Italy Goffredo et al.

A Table I. Vector status of the ‘C. obsoletus group’ in Italy, for Bluetongue 100% virus (BTV) and related orbiviruses (African horse sickness virus, AHSV, and Epizootic haemorrhagic disease virus, EHDV). 80% 20 13 26 26 25 26 12 Laboratory 15 30 14 30 Field 60% 174 34 38 38 conditions Association ‘Virus 40% Virus Oral with detected isolation infection 20 outbreaks by PCR’ 20% 7 14 14 15 14 8 5 10 6 10 26 6 C. chiopterus none - - - 2 2 0% C. dewulfi rare BTV-1 - - Jul-06 Jan-06Feb-06Mar-06Apr-06May-06Jun-06 Aug-06Sep-06Oct-06Nov-06Dec-06Jan-07Feb-07Mar-07 BTV AHSV-4; Obsoletus BTV-2; Month/year frequent (various AHSV-9; complex BTV-9 C. obsoletus C. scoticus Obsoletus complex serotypes) EHDV-6 B AHSV-4; 5 C. obsoletus frequent BTV-1 - AHSV-9; 4.5 EHDV-6 4 3.5 AHSV-4; 3 C. scoticus frequent BTV-1 - AHSV-9; 2.5 EHDV-6 2 Log midges Log 1.5 C. montanus rare BTV-1 - - 1 0.5 0 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 0 -0 -0 -0 0 0 0 0 0 0 0 -0 0 -0 -0 b r v- c b So far, the species of the Obsoletus complex have Jul- Di Jan- Fe Mar Ap May- Jun- Aug- Sep- Oct- No Jan- Fe Mar been involved in Italy in the transmission of BTV Month/year and of Schmallenberg virus (Goffredo et al. 2013, Figure 6. Seasonal abundance of C. obsoletus and C. scoticus in Balenghien et al. 2014, Goffredoet al. 2015). a permanent collection site in Central Italy, basing on 15 biggest In Italy, the Obsoletus complex has repeatedly been collection/month (January 2006 - March 2007). From each collection found PCR‑positive to BTV genome and representing at least 20 random nulliparous were identified by multiplex PCR A( ). multiple serotypes (BTV‑1, BTV‑2, BTV‑4, BTV‑8, The species abundance has been extrapolated to the total Obsoletus BTV‑9, BTV‑16); furthermore, two serotypes, namely complex of the collections (B). BTV‑2 and BTV‑9, were isolated successfully from this vector complex (Savini et al. 2005). Subsequently, single midge specimens, identified molecularly to of the two species (C. obsoletus s.s. and species and representing all three species of the C. scoticus) do not change across the year. Obsoletus complex, were found PCR‑positive for Culicoides scoticus was consistently the most BTV (Goffredoet al. 2015). abundant of the two species, its relative Finally, piecemeal observations (mostly abundances in collections ranging from 50% unpublished) made on the Obsoletus complex and in December to 95% in September (average of potential relevance to vector capacity and the 76.6%); for both species, abundances peaked overwintering of viruses in adult females, were made: in May‑June, but differed in that the peak in (i) midges are active during daylight hours, (ii) feed C. scoticus was more pronounced (Figure 6). on livestock inside stables, and (iii) in winter can be found flying at temperatures below 5°C. In addition, midges maintained under laboratory conditions Vector status without a blood meal, but fed a sucrose solution, can In Table 1 the vector status for each of the five survive for up to 104 days at 17‑25°C. Furthermore, species of the ‘C. obsoletus group’ in Italy is shown, refrigerated midges maintained without sustenance based on field infection rates involving BTV, and and light for 10 days at 4°C were able to recover fully laboratory infection rates for related Orbivirus and suddenly thereafter (Goffredoet al. 2004). (Goffredo et al. 2015, Federici et al. 2016). Of the five These findings prove that BTV might overwinter in species, only C. chiopterus has not been implicated C. scoticus and C. obsoletus adult females and the same in BT outbreaks; BTV‑1 was detected in parous can happen for related Orbivirus, such as African horse C. dewulfi for the first time in Italy in 2014. sickness or Epizootic haemorrhagic disease viruses.

240 Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1 Goffredoet al. ‘C. obsoletus group’ in Italy

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